Frozen Single Cell under Raman Spectroscopy

BPJ_112_12.c1.inddCryopreservation is the technology used to stabilize cells for a variety of applications, including diagnosis and treatment of disease. Because we don’t completely understand the mechanisms of freezing damage, poor or inadequate methods of preservation have limited our ability to use cells for cell therapy. Also, observations of cell responses could not be correlated to viability on a cell-by-cell basis using conventional low-temperature microscopy techniques. This study establishes our ability to measure the viability of individual frozen cells based on the correlation of cytochrome c distribution, a signal that can be detected using Raman spectroscopy, with trypan blue staining. With Raman spectroscopy, we are able to observe cells during freezing, and identify specific chemical and morphological changes inside the cell that result in life or death.

The cover image for the June 20 issue of the Biophysical Journal is an artistic rendering of frozen cells surrounded by extracellular ice and unfrozen solution. The background image is Lake Michigan in cold winter. Floating ice is separated by unfrozen water. The distribution pattern of the floating ice and unfrozen water is just like a frozen sample: ice crystals are separated by unfrozen concentrated solution. Schematic diagrams of frozen cells were imbedded in the background image to mimic a real frozen cell sample. In the diagram, the blue area represents unfrozen solution, the white area represents extracellular ice, the red line represents a region of cell membrane in close proximity to extracellular ice, and the black line represents a region of cell membrane in unfrozen solution between adjacent extracellular ice crystals. The schematic diagrams were precisely positioned in the background image such that the unfrozen solution in the diagram was co-located with unfrozen water in the background image and the extracellular ice crystals in the diagram were co-located with the floating ice in the background image. Our studies found that interactions between the cell membrane and extracellular ice resulted in intracellular ice formation (IIF), and increasing the distance between extracellular ice and cell membrane decreased the incidence of IIF.

Raman spectroscopy has enhanced our understanding of freezing damage. These studies can enable the development of new and improved cell preservation protocols and eventually improve the growth of cellular therapies and our ability to treat patients.

– Guanglin Yu, Yan Rou Yap, Katie Pollock, Allison Hubel

Biophysicists Finding Balance: Father’s Day 2017

June 18 is Father’s Day in the US. In honor of the occasion, we spoke with Biophysical Society member Seth Weinberg, Virginia Commonwealth University, about what it is like to be a biophysicist and a parent, and how the two roles impact each other.


How many children do you have? What are their ages?

My wife Gabrielle and I have 3-year old twin girls, Hannah and Meredith.

At what stage of your career did you have your children? 

My daughters were born about three months before I started my first faculty position.  It was a very hectic time, finishing up the last few months of a post-doc, moving, and then starting up my own research group.

 Has your career been influenced or changed by your role as a parent? How?

I like to think that my experiences raising twins has made me more patient with my own students. Having become a parent and starting my own lab at roughly the same time, I have thought more about my role in preparing the next generation.  Especially being the father of two daughters, I have become more aware of how important it is to promote opportunities for women in science.

How has your career been influenced by your own father?

My own scientific career has stemmed from my desire to understand how complicated systems work, and much of that desire originates from my father.  My father was a science and engineering teacher, and from a very young age, he encouraged me to make learning and being curious into activities that were fun.

What has been the most challenging aspect of being a biophysicist and a parent?

Being away from my family to attend meetings and conferences is definitely challenging.  As much as I enjoy seeing and catching up with colleagues, it is still difficult to be away from my family during trips.

Have there been any benefits to being both a father and a scientist?

In perhaps a subtle way, my daughters have inspired my interests as a scientist.  My daughters are fraternal twins, and since their birth, I’ve become more interested in ‘nature vs. nurture’ types of questions.  I’m constantly amazed at how different my daughters are, despite their nearly identical childhoods (so far at least).  As a scientist, I’ve become interested in understanding how important randomness is in our biology and trying to understand how one system can generate different behaviors in response to the same input.

Would you encourage your children to be scientists?

I don’t plan to encourage them to be scientists any more or less than other specific careers.  I hope to encourage them to pursue careers that they will enjoy and find fulfilling.  Although, with my own career and my wife as a nurse, I wouldn’t be surprised if one or both pursued some career in the biomedical sciences.

How would your children describe your work?

I asked my daughters what they think I do at work. One said, “Monkey!” and the other laughed.

Any advice for other fathers or prospective fathers pursuing science careers?

Pursuing a science career is challenging – constantly being pulled in multiple directions and never knowing for sure if you are doing things right.  Being a parent is pretty much the exact same thing, so as a scientist, you are as prepared as you can be (which is to say, you are never really prepared)!

Pre-Meeting Introduction: Single-Cell biophysics: measurement, modulation and modelling

Hi everyone. I’m Michael and I’ll be one of three people covering the meeting. I’m a single cell biophysicist from London, currently working in Dylan Owen’s lab at King’s College (KCL).

The lab and I work on ways to quantitate biological phenomena on the nanoscale, particularly molecular clustering. I personally have been looking into the nanoscale goings on of integrin adhesions in migrating T cells. It’s a fun system to work in, because the adhesions are so much smaller than what are found in most other cells. What I’ve found appears to be a specific system of membrane nanoclustering, that is altered to tune the speed of a migrating T cell.

We think that this might be pretty important, especially considering a speed change experienced by cells with a mutation in PTPN22 – an integrin signal modulating phosphatase that is associated with autoimmune disease predisposition.

As well as this, we are very interesting in the dynamics and ultrastructure of the actin cytoskeleton, and mechanisms of membrane protein nanoclustering – how this might relate to the picket-fence model, lipid rafts and scaffold type protein regulation.

Some very relevant topics await: on the first day I’m very interested to hear Suliana Manley for live super resolution microscopy and Pakorn Kanchanawong on his cadherin adhesome work. I hope you are too!

As light rain continues to fall over Taipei, I think I can hear some kind of horn (of Gondor?), which I’m going to take it as my signal to start exploring the city. I always enjoy the sounds you get in such a place – city sounds are my favourite as they are so complex and multi-layered – familiar somehow and yet so different to what I’m used to in London.

So, with that, I’ll see you all tomorrow, for an engaging first day full of imaging techniques, mechanobiology, nanotechnology and the cell cycle!

Michael Shannon (Dylan Owen lab, KCL)

Using Biophysics to Understand Cataracts

June is Cataract Awareness Month in the US. By age 80, more than half of all Americans either have a cataract or have had cataract surgery, according to the National Eye Institute. To recognize this month, we spoke with BPS member Doug Tobias, University of California, Irvine, about his research on crystallin proteins, the proper functioning of which is key to lens clarity. 

What is the connection between your research and cataracts?

The eye lens, which focuses light on the retina, is composed of bundled fiber cells that lose their nuclei, ribosomes, and organelles during embryonic development. The transparency and refractive properties required for proper lens function results from the liquid-like order of structural proteins called crystallins, which are present in these cells at concentrations exceeding 300 g/L. Because the central nucleus of the lens undergoes very little protein turnover, crystallin proteins must remain stably in solution for a lifetime. A cataract is an opacification of the eye lens caused by the loss of solubility of the crystallin proteins, leading to the formation of aggregates that scatter light.

Tobias image 2

All-atom model of AQP0 tetramer embedded in a hydrated lipid bilayer and complexed with two CaM molecules. Reference: S. L. Reichow, D. Clemens, J. A. Freites, K. L. Németh-Cahalan, M. Heyden, D. J. Tobias, J. E. Hall, and T. Gonen, Allosteric mechanism of water-channel gating by Ca2+–calmodulin, Nat. Struct. Molec. Biol. 20, 1085-1092 (2013).

Our research seeks to understand cataract formation on the molecular level from two different angles, both involving atomically-detailed computer simulations (molecular dynamics, Brownian dynamics, and Monte Carlo), in concert with experiments carried out by our collaborators. On one front, we are attempting to elucidate how minor changes to the chemical structure of the crystallin proteins – such as the single-point mutations that are associated with congenital, early onset cataract, or post-translational modifications (e.g., truncation, disulfide bond formation, UV damage, deamidation, etc.) in the case of the much more common age-related cataract – give rise to altered interprotein interactions, which, in turn, lead to protein aggregation.

On the other front, we are learning how the protein aquaporin 0 (AQP0) is regulated. AQP0 is expressed exclusively in the eye lens, where it comprises roughly half the membrane protein content and functions as a water channel and, hence, is a key player in maintaining lens homeostasis. AQP0’s water permeability is regulated by pH and Ca2+, whose concentrations change dramatically with distance into the lens. Defects in AQP0 can lead to cataract. We are using molecular dynamics simulations, in conjunction with experimental measurements carried out by our collaborators, to determine the mechanism of the alteration of AQP0 permeability by certain mutations, as well as by changes in pH and Ca2+ levels.

Why is your research important to those concerned about cataracts?

There is good reason to be concerned about cataracts. In its latest assessment of priority eye diseases, the World Health Organization estimated that cataract is responsible for more than 50% of world blindness, which amounts to roughly 20 million people. Presently, cataracts are treated by surgical replacement of the opaque lens with an artificial intraocular lens.  Due to lack of access to eye care, millions of people in the developing world remain blind from cataract. The long-term goal of our research is to gain new insights into cataract formation that could ultimately guide the development of new therapies for cataract prevention and treatment.

How did you get into this area of research?

I got into this area of research through interactions with colleagues at UC Irvine, specifically, Jim Hall and Rachel Martin. Jim Hall is a lens physiologist in the Department of Physiology and Biophysics and an expert on AQP0, and Rachel Martin is a biophysical chemist in the Department of Chemistry and an expert on the crystallins. Their enthusiasm for their respective subjects was infectious and irresistible, and I jumped at the opportunity to collaborate with them. Our collaboration with Jim Hall was initiated by a graduate student in his lab who wanted to spend time in my lab learning molecular modeling. Our work with Rachel Martin began as a summer undergraduate research project.

How long have you been working on it?

We started dabbling in the field about five years ago and then increased our effort substantially after receiving funding for the work about three years ago.

Do you receive public funding for this work? If so, from what agency?

Our work on the crystallin proteins is supported by both the National Institutes of Health and the National Science Foundation, and our work on AQP0 is supported by the National Institutes of Health.

Tobias image

Computational model of an aggregate formed by the congenital cataract-related W42R mutant of human gD-crystallin in a solution at 220 g/L concentration. The N-terminal domains are colored red and the C-terminal domains blue. The aggregates formed by the W42R mutant display enhanced interprotein contacts involving the N-terminal domain, where the mutation is located, vs. the wild-type protein, which displays primarily non-specific interactions at the same concentration.

Have you had any surprise findings thus far?

Ca2+ regulation of AQP0 is mediated by the calcium binding protein calmodulin (CaM), which forms a complex with AQP0 of unusual stoichiometry (2 CaM to 1 AQP0 tetramer). We expected that CaM binding would lower AQP0 permeability by simply blocking the water conducting pores. However, we found instead that CaM binding lowers the open probability of a gate deep within the pore through long-range allosteric interactions. We are now learning, also unexpectedly, that pH regulation occurs through a similar (allosteric) mechanism involving a second gate on the other end of the pore.

Crystallin proteins are constantly bumping into one another due to their high concentration in the lens. “Healthy” (wild type) crystallins stay in solution because they don’t stick to each other, i.e., their interactions are non-specific. It is fascinating to me that a single-point mutation in a single class of crystallin proteins can lead to early-onset cataract. The aggregation mechanisms of these mutant proteins are only beginning to be worked out, and it is likely that different mechanisms will be at play in different cataract-related variants. In one case that we are currently studying, we found that the W42R mutation “cracks open” the interface between the two domains in gD-crystallin, exposing a sticky patch that promotes the formation of large-scale protein clusters.

What is particularly interesting about the work from the perspective of other researchers?

Our work produces atomically-detailed descriptions of molecular mechanisms that are often critical to the interpretation of experimental observations. We take great pains to validate our simulations so that their predictions can be trusted.

What is particularly interesting about the work from the perspective of the public?

Cataract is the leading cause of blindness worldwide.  Even in developed countries like the US, where cataract surgery is widely available, and safe and effective in the vast majority of cases, delaying it for just a few years would greatly reduce health care costs. We hope that our research will yield new fundamental insights that will inform non-surgical approaches to cataract treatment.

A Dynamic Biochemomechanical Model of Geometry-Confined Cell Spreading

BPJ_112_11.c1.inddCell spreading is involved in many physiological and pathological processes. In confluent multicellular systems, the dynamic evolution of an individual cell is influenced by its neighbors. It has been recognized that microsystems (e.g., microchambers) with defined geometry can affect the spatiotemporal dynamics of cells. However, it remains unclear how cells sense and respond to geometric confinement at the subcellular level. We answer this question by establishing a dynamic biochemomechanical model of geometry-confined cell spreading. This model reveals that the positioning of the cell-division plane is strongly affected by its boundary confinement.

The cover image for the June 6 issue of the Biophysical Journal illustrates the dynamic configurational evolution of a cell (blue) spreading in an L-shaped microchamber (silver). Its nucleus and microtubules are represented by the green sphere and emanated lines, respectively. The cell flattens and forms lamellipodia on the substrate but cannot step over the side walls of the chamber. In the initial spreading stage, the cell takes a round shape and spreads isotropically on the substrate before contacting the chamber boundary. The nucleus is positioned at its mass center (remote). Once the cell membrane contacts the chamber boundary, it may slide along or be fixed on the latter, depending on the force equilibrium condition (middle). For a cell undergoing anisotropic spreading, the length-dependent microtubule forces can drive the nucleus to move. Finally, the spreading cell reaches a steady-state configuration, which dictates the nuclear deformation and the cell-division plane (near).

The cover image was inspired by the cell spreading dynamics model which integrates biological, chemical, and mechanical mechanisms based on experimental observations. More details of the model can be found in our Biophysical Journal paper. This interdisciplinary work helps understand how microenvironments affect the spreading dynamics and division of cells. The findings also have potential applications in regulating cell division and designing cell-based sensors.

– Zi-Long Zhao, Zong-Yuan Liu, Jing Du, Guang-Kui Xu, Xi-Qiao Feng

BPS Summer Research Program TA Spotlight: Mike Pablo

mike-pblo-IMG_2706-CR-desk-150-6The 2017 Biophysical Society Summer Research Program in Biophysics is currently underway at UNC Chapel Hill. We caught up with one of the program’s teaching assistants, Mike Pablo, to learn about his current research, how he became interested in biophysics, and what he’s looking forward to this summer. 

How did you get started in science in general and biophysics in particular?

For a time, I thought I would want to pursue medicine, but a stint volunteering with an ambulance corps in high school showed me otherwise. After that, I didn’t know what I wanted to study. When I eventually applied to colleges for my undergraduate degree, I recklessly submitted a different major for each application. I wound up studying Chemistry at Northeastern University thanks to a wonderful scholarship. While there, I was lucky to get involved with PRISM (“Proactive Recruitment In Science and Mathematics”), a program aimed at freshmen to get them interested in research problems. Both chemistry and research were interesting, so I stuck with it! Over the years, I found myself really enjoying both quantitative, mathematically-grounded work as well as biochemistry. This led me towards bioanalytical and analytical chemistry, and I didn’t break into any biophysical studies until I came to graduate school and got involved with the Training Program in Biophysics and Molecular Cell Biology, which was a fantastic experience.

Are other members of your family involved in science? If not, what sort of work were your parents or guardians involved in while you were growing up?

Nope! My mother is a nurse, and my father works in life insurance.

Where did you grow up?

I grew up in Queens, New York. Between living in New York and attending college in Boston, I’m used to big cities. Moving to Chapel Hill was a little challenging, but the place has grown on me a lot.

What schools have you attended/are you attending? What degrees do you hold?

I went to the Bronx High School of Science, then to Northeastern University, where I got a BS in Chemistry.

What is your current position? Please describe any current projects or research.

I’m now a PhD candidate in the Chemistry department at the University of North Carolina at Chapel Hill, more specifically within the Biological division. My current research uses computational approaches to understand how biochemical signaling is coordinated during phagocytosis, a process very fundamental to our immune systems. We know that many key molecules in phagocytosis need to be distributed within the cell in precisely shaped and timed ways: disrupting this coordination can lead to impaired or failed phagocytosis. So how do cells consistently manage it? I’m building both simulations of the biochemistry within the cell to get an understanding of how it works, and tools to analyze experimentally-acquired data.

Why did you want to be a TA for the BPS Summer Research Program? 

It sounded like a fun program to be a TA for, and I’m looking forward to helping our students become more proficient as researchers. I think it’s a great opportunity for students to experience research, and I know how valuable that opportunity can be from my PRISM experience and work I did during my undergraduate degree. The course has just barely started, but I look forward to seeing them grow over the next couple of months.

Name someone you admire and explain why.

I’m going to cheat a little and name several: Randall Munroe, Zach Weinersmith, and Kate Beaton. All of them use a fun medium (comics) to communicate information that people might normally find boring, ranging from science and engineering to philosophy and history. I think it’s an amazing way to spark interest, and believe they have a great positive impact on a wide range of people.

What are your future plans for your career/research?

I think I still want to pursue computational approaches to study complex biochemical and biological systems. It’s a little hard for me to nail down something more specific – I feel that there’s still so much more for me to explore in terms of what I could apply my training to.

Nothing is Impossible

BPJ_112_10.c1.inddRecent work in molecular bioelectricity has demonstrated the ability to radically alter animals’ morphology despite a normal genomic sequence. Cells make decisions and cooperate towards complex anatomical goal states using bioelectric gradients that are only detectable in the living state and invisible to the mainstream protein or mRNA profiling approaches. The study of these non-neural bioelectrical networks have allowed us to create living “impossible objects” in the highly regenerative planarian flatworm system. For example, flatworms can be made permanently two-headed by a transient change of their bioelectrical circuit. A brief shift of their bioelectric network to a new attractor state permanently alters their pattern memory so that in the future, they will regenerate as two-headed forms out of middle fragments cut in plain water, despite their wild-type genomic sequence.  M. C. Escher was an artist with a keen appreciation of “impossible” or “undecidable” objects, drawing many two-dimensional forms (such as ever-descending staircases) that cannot exist in our three-dimensional world.

We drew inspiration for our image on the cover of the May 23 issue of Biophysical Journal from Escher’s visions, as the worms we report in our paper are in a sense “impossible objects,” whose target morphology does not match their current anatomy. They are also “undecidable objects” because each worm stochastically decides to be one or two-headed upon amputation, persisting in an undecided state. The image is specifically based on the M. C. Escher woodcut Another World II, a.k.a. Other World II, which masterfully depicts paradoxical views of an alien landscape, revealing different aspects of reality but not matching our expectations based on the perspective we take looking through each of the windows in the structure.

ImageThis image is a perfect complement to our work on the physiological determinants of patterning. These experiments revealed a new perspective on the control of biological anatomy, which exhibit rules and properties quite different from what is seen when the same object is viewed through the portal of genetic networks or biochemical gradients. To adapt the original woodcut for this image, the color was changed to the blue/red pseudocoloring used to image voltage gradients in the planaria, and the bird-like creature Escher used in the original has been changed to one- or two-headed planaria.

Interestingly, Escher was well aware of the remarkable properties of planaria, as shown  in his 1959 lithograph Planaria (Flatworms). However, the study of bioelectric regulation of growth and form applies well beyond planaria.  It is relevant to the detection and repair of birth defects (especially of the face, brain, and left-right axis), the induction of regeneration of limbs, and detection or reprogramming of cancer, as well as synthetic bioengineering. Much like neural networks in the brain, somatic bioelectric networks store pattern memories and process information that guide development, regeneration, and cancer suppression. Beyond biomedical applications in regenerative medicine and bioengineering, the study of bioelectric communication within tissues in vivo is a branch of the emerging field of primitive cognition.

Evolution takes advantage of biophysical processes to drive computation and decision-making in the brain and body; learning to manipulate this process may allow us to achieve currently impossible biological objects, with structure and function far beyond those we can envision today.

The cover image was created by Jeremy Guay, of Peregrine Creative.

– Fallon Durant, Junji Morokuma, Christopher Fields, Katherine Williams, Dany Adams, Michael Levin